1. Field of the Invention
This invention relates to confocal microscopes which detect light reflected or emitted from a sample. This invention also relates to scanning confocal microscopes which provide high quality images at relatively low cost, and which can scan samples in at least one axis using a moving objective lens.
2. Background of the Invention
Confocal microscopes have been known at least in the literature since the 1950's. These microscopes use a visible originating radiation source (although some configurations may now use infrared IR! or ultraviolet UV! radiation) which is directed to a beam splitter. The beam splitter directs a portion of the radiation from the source to a collimating lens, which further directs the radiation towards an objective lens. The radiation passing through and focused by the objective lens converges on a focal point. When there is an object at or about the focal point, the focused radiation is reflected back through the objective lens and the collimation lens towards the beam splitter. A further reduced portion of the reflected radiation which has passed a second time through the objective lens and collimating lens passes through the beam splitter and is detected by a detector such as a semiconductor, photovoltaic sensor or large area detector. The radiation emitting source for the originating radiation may be any source which is capable of producing collimated radiation or which may be sufficiently collimated by the collimating lens to provide sufficient coherency to provide resolution to the image, consistency to the quality of the reflection from an object at the focal point and transmission to the detecting devices after the second pass through the beam splitter. Although these devices are of high quality and effectiveness in the marketplace, they are relatively expensive and have significant limitations in their utility which the present inventors have determined is at least in part a result of the weight and size of internal components.
The objective lens in commercial confocal microscopes have traditionally consisted of the higher cost and higher quality quartz lenses, which have significant mass (tending to be at least 20 grams and as much as 50 grams for the lens itself). The lens is moved relative to the target at the focal point by either gross movement of the microscope (with a fixed focal length), movement of the object target (again with a fixed focal length), by movement of the objective lens (changing the focal length), or combinations of these procedures. In those situations where the objective lens is to be moved, the control over the movement, and more importantly the identification of the amount of the movement and its position within the microscope system, is effected by a closed loop system of voltage regulation to coils or piezoelectric devices attached to the objective lens. Changes in the voltage/current to the coils causes them to move in a predetermined direction to shift the position of the objective lens. There are two or more coils (or sets of coils) attached to the objective lens to control movement of the lens in two or more axial directions. The two most important directions are 1) parallel to the incoming radiation from the columnating lens and 2) at least one of the two axes perpendicular to the direction of the radiation from the columnating lens. Direction 2) tends to be fixed (i.e., it is essentially parallel to the surface being scanned and can not readily move within the plane of lines or directions perpendicular to the radiation moving through the columnating lens) and is a single fixed direction often defined as the scanning direction relative to the object or target. The position of the focal point and the objective lens is estimated by the current/voltage provided to the coils. This has been sufficient for the accuracy needed in the confocal microscopes, but is not stable in its realistic accuracy. The response of the coils changes with time, the accuracy of the voltage/current readings change with time, the initial position of the objective lens may shift from impact or vibration, and other physical changes in the system alter the performance and accuracy of the determination of the position of the objective lens and focal point. In many systems, this is not necessarily a problem, as where in profilometers it is the relative variations in the surface which are important and not necessarily the actual position on a surface which is the primary interest of the observer.
In simple terminology, confocal microscopes use a detection method that preferentially measures light that is emitted (e.g., by phosphorescence or fluorescence) or reflected near the focal point of a beam of light. This is typically done by detecting reflected light which is returned through the same objective lens that originally projects a light beam at a target or object and subsequently measuring a portion of the returned light which follows or retraces a portion of the beam path of the illumination source after it has passed through a beam splitter. Microscopic images can be created by scanning either the beam or the sample through two or three axes while measuring signal intensity. Confocal detection offers greatly improved vertical resolution and clarity compared to conventional microscopy. The limitations of confocal microscopy are that the image is acquired by physically moving the focal point over or through a sample, which is generally a slow and often an optically complex process when compared to conventional optical microscopy. Much of the complexity in a confocal microscope is in moving the focal point. Moving the focal point by scanning the light beam can be accomplished either by moving pinholes or by deflecting the beam before the objective lens. Confocal microscopes based on scanned pinholes or aperture are commercially available. These types of confocal microscopes are readily adapted to use much of the optics in a conventional optical microscope. An alternative approach that achieves the same result is to fix the pinhole and scan the beam, illuminating the object or target in an arc, pattern or line form within one plane or two dimensions. Both the scanned beam and the scanned pinhole approaches require expensive objective lenses to provide a wide field of view with near diffraction limited resolution. In many cases, these confocal microscopes also preferably use expensive polarization preserving objective lenses to provide high signal throughput.
Another approach for achieving a scanned focal point in a sample is to scan the object or target co-linearly with the beam of light while the beam and pinhole are fixed in at least one scan axis. The other axes of motion are provided by either moving the sample, or the optical assembly, or both. This type of approach is exemplified in U.S. Pat. Nos. 5,179,276 and 4,863,252. All degrees of motion can also be provided through either the optical assembly, the sample, or a combination of the two. The latter two approaches have limited utility due to the mechanics of rapidly and accurately moving the samples and optical assemblies to provide two or three dimensional images. Resonant scanning of the objective can be used in some devices, although resonant systems are usually at a fixed frequency and are limited in their ability to pan over large sample areas.
The above approaches to confocal microscopy are typified as being inherently bulky and complex.
Italian Patent No. 1203297, published on Feb. 16, 1989 describes a profilometer for measuring the profile of a surface using a confocal distribution of optical assemblies (e.g., radiation source 3, collecting lens 8, beam splitter 5, spatial filter 9, and photoconverter 6). The position detector 14 for determining the instantaneous position of the lens 4 merely indicates electrical signals sent to a piezoelectric device 25a and 25b to oscillate the lens 4. The signal may be indicated by a potentiometer 27 with an indicator needle 27a. There is no collimating lens before the beam splitter, the lens oscillates to create movement of the focal point, single direction variation of the lens is provided (FIG. 2, device 12 for moving lens 4 parallel to its optical axis.
Keyence Corporation of America, 50 Tice Blvd., Woodcliff Lake, N.J. 07675 markets a displacement metering device LT-8110 laser displacement meter noteworthy for its long working distance in the operation of its focal point. The device has a light emitter, beam splitter, objective lens and finishing optical unit (in that sequence in the operation of the microscope). Light reflected off the target passes back through the finishing optics and the objective lens, to be broken into two paths by the beam splitter. One path continues essentially linearly to a light detection unit (e.g., photodetector) and the other path is deflected to another sensing unit whose operation is not understood. The working distance for the focal point is between about 10 and 28 mm, which would be quite large for a confocal microscope. This large working distance appears to be possible because of a relatively large focal length for the objective lens as compared to the diameter of the objective lens. This means that the effective F-number of the system is large (e.g., about 2). Lens systems with large F-numbers have larger spot sizes (when focusing emitted radiation) and larger depths of field. Larger spot sizes tend to decrease the resolving power of the system, which is consistent with the Keyence device advertised as providing about a 2 micron diameter laser beam (effectively defining the resolution of the system as comparable to about 2 microns). The Keyence microscope also is shown in the literature to have the objective move in only a single axis (the axis parallel to the laser beam). It is possible that the lens is moved in an arc, but this is still single direction control, without the capability of independent movement along at least two axes. The finishing optical unit appears to enhance the large working distance in this microscope by adjusting the angle of the radiation after it has passed through the objective lens.